Structural Role of Flightin in Thick Filaments of Drosophila Flight Muscle

智力运动是生物体的基本特性之一。在大多数动物中，运动是由横纹肌的收缩提供动力的，之所以这样称呼，是因为它们的蛋白质细丝高度规则和重复的组织，在显微镜下可以看到条纹。所有的横纹肌都含有粗纤维，其主要成分是肌球蛋白，一种进化上保守的运动蛋白，负责产生肌肉收缩的力量。来自不同肌肉类型的粗丝具有不同的结构和机械特性，这些特性是由非肌球蛋白粗丝蛋白赋予的。因此，这些蛋白质在确定肌肉类型之间的功能差异方面起着重要作用。这项研究将解决飞行蛋白的功能，飞行蛋白是一种新型的厚丝蛋白，对果蝇飞行肌肉的结构完整性和机械稳定性至关重要。具体来说，本项目旨在通过体外生化研究和体内遗传研究相结合的方法来阐明飞行蛋白和肌球蛋白之间的分子相互作用。这项工作的一个关键假设是，飞行蛋白中的一个保守氨基酸区域（大约有52个氨基酸）确定了一个新的肌球蛋白结合区域，该区域广泛存在于昆虫和甲壳类两大无脊椎动物群体中。为了阐明飞行功能的结构基础，将利用电子显微镜获得的图像，通过断层扫描生成果蝇飞行肌粗丝的详细三维模型。结合分子标记技术，比较有和没有飞行蛋白的纤维结构，可以在粗纤维中识别和定位飞行蛋白。这些研究将提供第一个来自模型遗传生物的高分辨率的粗丝三维结构，并将为大量具有已知遗传、生化和机械缺陷的果蝇飞行肌肉突变体的结构分析提供基础。此外，这项研究的影响将扩展到进化生物学和系统发育学领域，这些领域试图理解以昆虫为例的生命形式的多样化，昆虫可以说是最成功的动物群体。更广泛的影响这项研究将对果蝇和肌肉研究社区的教育和生物试剂的产生产生影响。美国国家科学基金会此前对该研究小组的资助有助于培养本科生和研究生，包括来自弱势群体和弱势背景的成员，该项目旨在将研究与继续教育机会结合起来。本研究产生的知识将用于高级本科和研究生专业课程、针对研究社区的评论文章和书籍章节，以及针对非科学受众的文章和故事。研究结果将通过我们的网站、同行评议期刊的出版物以及将在科学会议上展示其研究成果的本科生和研究生广泛传播。所有试剂（DNA克隆，突变和转基因菌株）将免费提供给其他研究人员。

Intellectual MeritMovement is one of the fundamental properties of living organisms. In most animals, movement is powered by contraction of striated muscles, so called because of their highly regular and repetitive organization of protein filaments that are seen as striations under a microscope. All striated muscles contain thick filaments whose main component is myosin, the evolutionarily conserved motor protein responsible for generating the force for muscle contraction. Thick filaments from different muscle types have distinct structural and mechanical properties that are imparted by non-myosin thick filament proteins. Thus, these proteins play a significant role in defining functional differences among muscle types. This research will address the functions of flightin, a novel thick filament protein that is essential for the structural integrity and mechanical stability of the flight muscle in the fruit fly, Drosophila melanogaster. Specifically, this project aims to elucidate the molecular interaction between flightin and myosin through a combination of in vitro biochemical studies and in vivo genetic approaches. A key hypothesis of this work is that a conserved amino acid region in flightin (of approximately 52 amino acids) identifies a new myosin binding domain that is widespread among two major groups of invertebrates, the insects and the crustaceans. To elucidate the structural basis of flightin function, a detailed three dimensional model of the thick filament from the fruit fly flight muscle will be generated by tomography from images obtained with an electron microscope. A combination of molecular labeling techniques and comparison of filament structures with and without flightin will result in the identification and localization of flightin in the thick filament. These studies will provide the first higher resolution three-dimensional structure of thick filaments from a model genetic organism and will provide the foundation for the structural analysis of a plethora of Drosophila melanogaster flight muscle mutants with known genetic, biochemical, and mechanical defects. Additionally, the impact of this research will extend to areas of evolutionary biology and phylogenetics that seek to understand the diversification of life forms as exemplified by the insects, arguably the most successful group of animals.Broader impactsThis research will have impacts in the areas of education and the generation of biological reagents for the Drosophila and muscle research communities. Previous NSF funding to this research group has been instrumental in training undergraduate and graduate students, including members from underrepresented groups and disadvantaged backgrounds, and this project is designed to integrate research with continuing educational opportunities. Knowledge generated from this research will find its way to advanced undergraduate and graduate specialty courses, review articles and book chapters intended for the research community, and articles and stories for the non-scientific audience. Results will be broadly disseminated through our website, publications in peer-reviewed journals, and by undergraduate and graduate students who will present their work at scientific meetings. All reagents (DNA clones, mutant and transgenic strains) will be freely available to other investigators.